Hepatic stellate cells (HSCs) undergo transdifferentiation into hepatic myofibroblastic cells (HMs) to participate in liver fibrogenesis. We demonstrated that this transdifferentiation is caused by loss of transcriptional regulation essential to adipogenesis. Treatment with the adipocyte differentiation cocktail or expression of a key adipogenic transcription factor such as PPARgamma, reverses a culture-induced HM phenotype to quiescent HSCs. These results identify PPARgamma as a therapeutic target and also highlight an analogy between preadipocyte-adipocyte differentiation and HSC transdifferentiation. Emerging evidence points to the importance of epigenetic regulation in adipocyte differentiation. Preadipocytic fibroblasts have increased expression of histone deacetylases (HDACs) and their recruitment to a promoter of ppar-gamma, deacetylated histones, and consequent repression of ppar-gamma. As adipocyte differentiation ensues, this negative regulation is lifted. We hypothesize that repression of ppar-gamma in HSC-HM transdifferentiation is caused by a similar epigenetic mechanism. In support, we show that the DNA methyltransferase inhibitor 5- azadeoxycytidine, that reduces methyl-CpG and HDAC recruitment, restores PPARgamma expression and HSC quiescence. Further, HSC-HM transdifferentiation causes increased expression of MeCP2, the methyl-CpG binding protein (MBP) that mediates HDAC recruitment. To test and extend our hypothesis to alcoholic liver fibrosis (ALF), we wish to determine: 1) whether in vitro HSC-HM transdifferentiation is caused by epigenetic repression of ppar-gamma; 2) whether HMs from experimental ALF exhibit similar negative epigenetic changes; and 3) whether knockdown of MeCP2 prevents HSC-HM transdifferentiation and ALF. To pursue the aims, we will collaborate with the laboratory of Derek Mann who brings in expertise in epigenetic regulation. DNA methylation will be analyzed for ppar-gamma gene. Expression of epigenetic regulators (HDACs, MBPs, HMTs) and their recruitment to the ppary promoter, will be assessed by immunoblot and chromatin immunoprecipitation (ChIP) assays. ChIP will also determine modifications of histones bound to the promoter. HCSs/HMs will be isolated from rats with intragastric ethanol infusion plus enteral LPS administration that produce accentuated ALF. Epigenetic analysis of these cells will be compared to that of HMs captured by laser micro-dissection of frozen sections. To assess the role of MeCP2, three complementary approaches will be used: HSCs isolated from MeCP2 deficient mice; siRNA knockdown of MeCP2 in LX2 human HSC line; and a Cre-lox adenoviral vector system to express MeCP2 shRNA in a HM-specific manner in vivo by Cre recombinase expressed under the control of a2(l) collagen enhancer in collaboration with Dr. Kazuo Ikeda, a developer of this system. This exploratory study will be first to disclose epigenetic regulation of HSC transdifferentiation in ALF. [unreadable] [unreadable] [unreadable]